Cationic liposomes for gene transfer

Abstract

The present invention relates to synthetic cationic lipids, liposome formulations and the use of such compounds to introduce functional bioactive agents into cultured cells.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a divisional of and claims the benefit of, U.S. application Ser. No. 09 / 447,688, filed Nov. 23, 1999, now U.S. Pat. No. 6,656,498. U.S. application Ser. No. 09 / 447,688 claims the benefit of U.S. Provisional Application Nos. 60 / 109,950, filed Nov. 25, 1998 and 60 / 110,970, filed Dec. 4, 1998. U.S. Application Nos. 09 / 447,688, 60 / 109,950, and 60 / 110,970 are each incorporated herein by this reference in their entireties.STATEMENT OF GOVERNMENT INTEREST[0002]This invention was made with government support under Grant Nos. NIH 5 P50 HL 19153, NIH 5 RO1 HL 45151 and NIH 5 RO1 AI 31900 awarded by the National Institutes of Health to Vanderbilt University. The U.S. Government has certain rights in the invention.FIELD OF THE INVENTION[0003]This invention relates to synthetic cationic lipid compounds, liposome formulations and the use of such compounds via lipofection to introduce functional bioactive agents / biologically active s...

AI Technical Summary

Benefits of technology

[0035]The LADOP cationic lipid is a very efficient molecule as a result of careful design. The overall structure offers a bilayer-forming lipid with good membrane fluidity. Therefore, the novel lipid can actively transfer genes without a commonly required helper lipid, DOPE. LADOP has a stable amide bond linkage between the head group and the lipid anchor to ensure stability in solution. It has two cationic charges, while lysl-PE has only one net charge per molecule. The diester bonds in LADOP render the lipid biodegradable. This feature has been demonstrated in our in vivo studies in which the animals tolerated repeated intravenous doses of DNA / LADOP complex well and transgene expression patterns of the first injection and second injection shortly after were identical both in magnitude and duration. In two recent reports, a twenty day interval between treatments was needed for effective repeat administration, using DOTMA and DOTAP, two formulations that have been widely used. The fact that the novel LADOP cationic compound mediated efficient transfection both intravenously and intratracheally, together with features such as simplicity of synthesis and formulation and minimal toxicity, make this a very attractive and useful reagent for in vivo gene delivery, and ultimately, gene therapy.

[0040]These new cationic lipids are micelle-forming lipids in their salt form and have excellent DNA condensation abilities and capability of forming small complexes with DNA. In vitro test revealed that most of these lipids have high transfection activity on cells in general. However, the two lipids belonging to group three, with three to five lysine groups symmetrically and spaciously distributed over the molecules are 2 to 5 times more potent than those lipids with similar numbers of lysine groups distributed in dendritic (tree) shape or linear pattern. These novel compounds clearly demonstrate the important relationship of structure and transfection activity of a cationic lipid.

Problems solved by technology

Although lipofection is quite efficient in vitro under serum-free conditions, its use in vivo when delivered as DNA / lipid complex, lipoples, via intravenous or airway routes, was limited to the presence of proteins and polysaccharides in the body fluids and mucus that strongly inhibit the transfection efficiency.

However, the improvements were also associated with noticeable toxicity of varying degrees of lipid / DNA treated animals due to toxic effect of large excess of cationic lipids or lipoplexes themselves.

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